ABSTRACT Mechanistic basis of lung dysfunction after RBC transfusion Transfusion of red blood cells (RBCs) is associated with excess mortality and pulmonary morbidity in anemic patients with critical illness or acute coronary syndromes, but the underlying mechanisms are poorly understood. Impaired oxygenation of blood by the lung is relatively frequent after transfusion. But little is known of how storage lesions of the RBC may contribute to adverse pulmonary and other outcomes. The recent discovery of an active role of the RBC in regulating blood flow -- a major determinant of its own function, O2 delivery -- in the lung and periphery, prompts a reexamination of the risk-benefit balance in RBC transfusion. RBCs release vasodilator S-nitrosothiols (SNOs) upon physiological exposure to hypoxia, contributing to O2-dependent blood flow regulation. The result is hypoxic vasodilation peripherally, and modulation of hypoxic vasoconstriction in the lung. Yet RBC SNO appears to optimize O2 uptake in the lung rather than compromising it. Preliminary Studies in stored human RBCs show deficiency in SNOs, and in RBC-dependent regulation of adaptive vascular responses in the lung in vitro and in vivo. In parallel there is storage-induced, progressive impairment in RBC deformability, which may also be governed by (loss of) SNOs. The project's central hypothesis is that storage-induced depletion of RBC SNOs leads to dysregulation of the flow of blood in the lung, compromising O2 uptake. To test this novel hypothesis, we will accomplish these Specific Aims: 1) Determine the role of (S)NO loss in the abnormal flow of stored RBCs in the lung. 2) Determine whether pulmonary flow of RBCs and O2 uptake after transfusion are improved by SNO repletion after storage of RBCs, or prevention of the loss of SNOs. Strategies will be tested that a) prevent depletion of SNOs by modifying current blood-banking practices; b) replete SNOs in stored RBCs ex vivo or in vivo. Biochemical, genetic and pharmacological approaches are used to achieve these Aims in human and mouse RBCs, isolated perfused mouse lungs, and intact mice. The results of these studies are expected to inform the rational design of therapies to improve the efficacy and outcomes of RBC transfusion in critically ill patients with anemia. Potential Impact on Veterans' Health Care Anemia is strongly linked to increased morbidity and mortality in Veterans. RBC transfusion for anemia is among the therapies most frequently administered to hospitalized Veterans. But surprisingly, RBC transfusion is associated in some patient groups with an increased risk of death and adverse pulmonary outcomes. The results of these studies should lead to improved methods for RBC processing, storage, and transfusion, providing particular benefits for older and more medically complex patients, such as acutely ill Veterans.